The Psp system of Mycobacterium tuberculosis integrates envelope

Molecular Microbiology (2015) ■
doi:10.1111/mmi.13037
The Psp system of Mycobacterium tuberculosis integrates
envelope stress-sensing and envelope-preserving functions
Pratik Datta,1 Janani Ravi,1 Valentina Guerrini,1
Rinki Chauhan,1 Matthew B. Neiditch,2
Scarlet S. Shell,3 Sarah M. Fortune,4
Baris Hancioglu,5 Oleg A. Igoshin5 and
Maria Laura Gennaro1*
1
Public Health Research Institute and 2Department of
Microbiology, Biochemistry, and Molecular Genetics,
New Jersey Medical School, Rutgers, The State
University of New Jersey, Newark, NJ 07103, USA.
3
Department of Biology and Biotechnology, Worcester
Polytechnic Institute, Worcester, MA 01605, USA.
4
Department of Immunology and Infectious Diseases,
Harvard T. H. Chan School of Public Health, Boston,
MA 021142, USA.
5
Department of Bioengineering, Rice University,
Houston, TX 77005, USA.
Summary
The bacterial envelope integrates essential stresssensing and adaptive functions; thus, envelopepreserving functions are important for survival. In
Gram-negative bacteria, envelope integrity during
stress is maintained by the multi-gene Psp response.
Mycobacterium tuberculosis was thought to lack the
Psp system since it encodes only pspA and no other
psp ortholog. Intriguingly, pspA maps downstream
from clgR, which encodes a transcription factor regulated by the MprAB-σE envelope-stress-signaling
system. clgR inactivation lowered ATP concentration
during stress and protonophore treatment-induced
clgR-pspA expression, suggesting that these genes
express Psp-like functions. We identified a four-gene
set – clgR, pspA (rv2744c), rv2743c, rv2742c – that is
regulated by clgR and in turn regulates ClgR activity.
Regulatory and protein–protein interactions within the
set and a requirement of the four genes for functions
associated with envelope integrity and surface-stress
tolerance indicate that a Psp-like system has evolved
in mycobacteria. Among Actinobacteria, the four-gene
module occurred only in tuberculous mycobacteria
Accepted 18 April, 2015. *For correspondence. E-mail
marila.gennaro@rutgers.edu; Tel. (+1) 973 854 3210; Fax 973-8543101.
© 2015 John Wiley & Sons Ltd
and was required for intramacrophage growth, suggesting links between its function and mycobacterial
virulence. Additionally, the four-gene module was
required for MprAB-σE stress-signaling activity. The
positive feedback between envelope-stress-sensing
and envelope-preserving functions allows sustained
responses to multiple, envelope-perturbing signals
during chronic infection, making the system uniquely
suited to tuberculosis pathogenesis.
Introduction
The intracellular pathogen Mycobacterium tuberculosis
responds to host-generated stress by extensive metabolic
remodeling that leads to growth arrest and reduced susceptibility to host defenses and most antimicrobial agents.
The inability of the host immune response to eliminate the
pathogen results in latent infection and billions of asymptomatically infected individuals (WHO, 2013). This huge
reservoir of infected humans poses a serious public health
threat since weakening of host defenses leads to development of pulmonary disease and transmission of infection.
Understanding mechanisms used by M. tuberculosis to
sense and adapt to host-mediated stress is crucial for
developing highly effective anti-tuberculosis strategies.
Stress-signal sensing typically occurs at the surface of
pathogenic bacteria (Rowley et al., 2006). With M. tuberculosis, the cell surface serves both sensing and
adaptive functions. For example, stress-sensing functions
include two-component systems, anti-sigma factors
and membrane-bound serine/threonine protein kinases
(Rodrigue et al., 2006; Alber, 2009; Bretl et al., 2011).
Adaptive functions include structural remodeling of the
bacterial envelope that alters signaling to macrophages
and presumably leads to intracellular conditions favoring mycobacterial dormancy (Davidson et al., 1982;
Cunningham and Spreadbury, 1998; Jain et al., 2007; Shui
et al., 2007; Lavollay et al., 2008). Maintenance of envelope integrity may be essential for proper expression and
integration of bacillary stress responses.
A cornerstone of the surface stress response of
M. tuberculosis is the genetic network centered on the
MprAB two-component system and the accessory sigma
factor E (σE), two transcriptional regulators that activate
each other (Manganelli and Provvedi, 2010; Tiwari et al.,
2 P. Datta et al. ■
2010). Sensing of stress occurs through MprAB, which
may be activated by protein misfolding in the extracytoplasmic space (Bretl et al., 2014), and through σE, which
is activated by stress-induced degradation of the phosphorylated form of its cognate anti-sigma factor (Barik
et al., 2010). How sensing of envelope stress by the
mprAB-sigE network translates into preservation of cell
envelope integrity is unknown.
The mprAB-sigE regulon includes the transcription regulator clgR (rv2745c) and the adjacent rv2744c (Manganelli
et al., 2001; Estorninho et al., 2010), a pspA homolog
(Provvedi et al., 2009). The function of M. tuberculosis
pspA is unknown, but in Gram-negative organisms, pspA is
a member of the envelope-stress-responsive, multi-gene
Phage shock protein (Psp) response system that mitigates
envelope damage by preventing proton leakage across a
damaged membrane, thereby maintaining proton motive
force (PMF; Darwin, 2005; Darwin, 2013; Joly et al., 2010).
In that system, PspA has a dual role: It is a stoichiometric
inhibitor of the cognate PspF regulator, and it is an
envelope-stabilizing effector as a component of an
envelope-bound, multiprotein complex comprising PspA
and the integral membrane proteins PspB and PspC
(Darwin, 2005; 2013; Joly et al., 2010). While the presence
of a pspA homolog does not imply the presence of a
complete Psp system, particularly in Gram-positive organisms (Joly et al., 2010), we were intrigued by the possibility
that M. tuberculosis might possess a functional Psp
system connected to the mprAB-sigE-clgR stresssignaling network. Such a connection would complete the
logical need for stress signaling to help the bacteria
recover from envelope damage.
In the present work, we used gene expression profiling,
mutant analyses and protein–protein interaction studies to
characterize operon structure and regulatory interactions
among clgR, pspA and two downstream genes (rv2743c
and rv2742c), and to assess the requirement for these
genes in physiological processes typically associated with
envelope integrity. We report that the four-gene module of
M. tuberculosis is found only in tuberculous mycobacteria
among Actinobacteria and that it exhibits regulatory and
functional characteristics similar to those seen with the
Psp system of Gram-negative bacteria. Since three of four
proteins in the M. tuberculosis module share no amino
acid sequence homology with their Gram-negative counterparts, our work reveals a novel example of parallel or
convergent evolution in bacteria of different phyla. Moreover, the four-gene module and the mprAB-sigE network
are connected by a positive feedback circuit. Reciprocal
positive regulation of surface-stress signaling and envelope maintenance functions could enable tubercle bacilli
to respond to multiple surface-perturbing challenges from
the macrophage during the course of infection and therefore have an important role in mycobacterial virulence.
Fig. 1. A. Effect of clgR deletion on intracellular ATP levels.
Mid-log cultures of wild-type and clgR mutant strains were treated
with 0.03% SDS for 24 h. Extracts from pre and post-treatment
cultures were then assayed for ATP levels and expressed as ng of
ATP per mg of total protein.
B. Effect of treatment with carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) on expression of clgR and pspA. Mid-log
cultures of the wild-type strain were treated for 6 h with various
concentrations of CCCP, as indicated, and harvested pre- and
post-treatment. Total RNA was extracted, and amplicons were
measured by quantitative RT-PCR using primers and gene-specific
molecular beacons. Amplicon copy numbers were normalized to
16S rRNA. 0 μM = solvent-only control (corresponding to the
highest solvent concentration used with the CCCP treatment). In
this and subsequent figures, quantitative RT-PCR data are
presented as mean values (± standard error of the mean) from
triplicate experiments.
Results
Relationship of clgR-pspA with energy generation
and PMF
During envelope stress, the Psp regulon of Gram-negative
bacteria affects cellular activities that depend on PMF and
ATP level (Darwin, 2005; 2013; Joly et al., 2010). To determine whether a putative clgR-pspA system has a similar
role in M. tuberculosis, we measured levels of intracellular
ATP in M. tuberculosis cultures stressed with bacteriostatic
concentrations (0.03%) of sodium dodecyl sulfate (SDS)
(see Methods). Two strains were used: a wild-type strain
and a clgR deletion mutant, obtained by replacement of the
clgR coding sequence with an antibiotic resistance cassette (Estorninho et al., 2010). While no difference was
observed between the two strains in the absence of stress,
the ATP levels in SDS-treated clgR mutant cultures were
one-third those obtained with similarly stressed wild-type
cells (Fig. 1A).
Energy generation correlates with maintaining PMF,
and dissipation of PMF caused by treatment with proton
ionophores, such as carbonyl cyanide m-chlorophenyl
hydrazone (CCCP), induces expression of pspA and its
product in Escherichia coli and Salmonella typhimurium
(Weiner and Model, 1994; Becker et al., 2005). When
we examined the effect of CCCP treatment on the
expression of clgR-pspA of M. tuberculosis, we observed
CCCP-concentration-dependent induction of both genes
(Fig. 1B). The lowered ATP concentration seen with the
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 3
129bp
A
mRNA (x10 4)
C
6
B
pspA
IG2
71bp
IG3
rv2743c
clgR
WT
rv2742c
compl.
4
2
0
pspA
pspA
rv2743c
rv2743c
D
12
clgR
IG1
IG2
IG3
8
4
0
mRNA (x10 4)
mRNA (fold change)
PclgR
18bp
IG1
clgR
12
60
No stress (0min)
Stress (90min)
8
4
0
0
rv2742c
rv2742c
120 180 240
WT clgR
P
cl gR
PclgR
Time (min)
E
ECF sigma factor
ECF sigma factor
TSS
Upstream
clgR
GATTGCGGGAACCAACCCCACCGCCGGCGGCGTTCACCTGAT G
SigA
TSS
Upstream
pspA
ATGGGTGGGGCAATGCGCGCTGACCCGATAAATTGAGCGAC A
IG1clgR
WT
IG1
Start codon
ATG
Start codon
ATG
clgR mutant and the CCCP-mediated upregulation of
clgR-pspA suggests that M. tuberculosis possesses an
active Psp response.
Regulation of the clgR-pspA-rv2743c-rv2742c region
by ClgR
We next examined the regulatory structure of the genomic
region encoding clgR-pspA. Downstream of pspA are two
additional genes (rv2743c and rv2742c) that are transcribed in the same direction and that are separated
from pspA and from each other by short (∼ 20 to ∼ 70 bp)
intergenic regions (Fig. 2A). Since clgR, pspA and
rv2743c were previously reported to respond to surface
stress in a sigE-dependent manner in microarray experiments (Manganelli et al., 2001), and the DNA upstream
from clgR contains ClgR-binding sites (Estorninho et al.,
2010), we first asked whether the stress response of this
gene set was clgR-dependent. By using quantitative
RT-PCR for transcript enumeration, we found that the
surface stress (0.03% SDS) response of pspA, rv2743c
and rv2742c was abrogated in the clgR deletion strain
(Fig. 2B). Mutant complementation with clgR restored
35–40% of the downstream gene expression (Fig. 2B),
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
Fig. 2. Structure and regulation of genes in the
clgR-pspA-rv2743c-rv2742c region.
A. Schematic representation of the region. Arrows represent
direction of transcription. The arrow PclgR indicates sequences
upstream of clgR expressing promoter activity. IG = intergenic
regions of indicated length, in bp.
B. Effect of clgR deletion on gene stress response. Mid-log cultures
of wild-type, clgR deletion mutant and complemented strains were
treated with 0.03% SDS and harvested pre-treatment (time 0) and
post-treatment (90 min). Amplicons were generated from total RNA
by quantitative RT-PCR using primers and gene-specific molecular
beacons from intragenic sequences for each gene, as indicated.
Amplicon copy numbers, normalized to 16S rRNA, are presented
as fold-change relative to time 0. Mean values (± standard error of
the mean) were obtained from triplicate experiments.
C. Enumeration of intergenic-sequence-containing amplicons.
Mid-log cultures of the wild-type strain were treated with SDS, and
samples were harvested pretreatment and at multiple times
post-treatment. Amplicons were generated from sequences internal
to clgR (reference gene) and from intergenic sequences along the
region, as indicated (see panel A). Data obtained from triplicate
experiments were normalized to 16S rRNA. (Note that copy
numbers of RT-PCR amplicons obtained with different primer sets
and molecular beacons cannot be compared with each other due to
variations in primer-associated efficiency of PCR amplification and
molecular-beacon-dependent detection sensitivity).
D. Analysis of promoter probe constructs. Two promoter::lacZ
fusions were generated with nucleotide sequences comprising
either 382 bp upstream of clgR (PclgR, see panel A) or 250 bp
upstream of pspA (IG1, see panel A) in wild-type and clgR mutant
strains. lacZ transcripts were enumerated by quantitative RT-PCR
prior to and 90 min following SDS treatment of mid-log cultures.
Data obtained from triplicate experiments were normalized to 16S
rRNA.
E. Promoter mapping upstream of clgR and pspA. The panel
shows nucleotide sequences upstream of clgR and upstream of
pspA (intergenic region IG1). Indicated in red are the two
transcription start sites (TSS) mapped in this work using the
genome-wide mapping method described in Methods. The TSS in
the top row had a ratio of converted to unconverted 5′ ends > 1.74
(adjusted P ≤ 0.01 for probability of being a processed 5′ end). This
TSS is preceded by -10 and -35 sequences (shown in blue)
characteristic of the extracytoplasmic function (ECF) sigma factor
SigE (Manganelli et al., 2001). The 5′ end in the bottom row had a
converted/nonconverted ratio of 1.58 and was confirmed to be a
TSS based on the presence of a very strong SigA -10 motif (in
blue) (Feklistov and Darst, 2011; Cortes et al., 2013). In both rows,
the annotated first codon (ATG) is as reported in the
M. tuberculosis genome sequence (http://tuberculist.epfl.ch/). nt,
nucleotides.
indicating that expression of each of the three downstream genes required a functional clgR. Additionally,
since all four genes exhibited similar stress response
profiles (Fig. S1), and pair-wise co-transcription of clgRpspA and pspA-rv2743c was previously reported with
unstressed M. tuberculosis cultures (Roback et al., 2007),
we tested the possibility of co-transcription within the fourgene set under surface stress. To do so, we used RT-PCR
to determine whether RNA was generated from the intergenic regions (Fig. 2A) under the surface stress conditions applied in Fig. 2B. We found that not only were
amplicons generated from the intergenic regions but also
that these amplicons exhibited stress-response expression profiles similar to that of clgR (Fig. 2C) and of the
4 P. Datta et al. ■
downstream genes (Fig. S1). This result strongly suggests the presence of one or more multicistronic mRNA(s)
encoded by the four-gene region. To further explore the
regulatory structure of the region, we used promoterprobe technology and constructed lacZ reporter fusions
with DNA sequences upstream from clgR and within
the clgR-pspA intergenic region (IG1 in Fig. 2A). When
reporter gene expression was measured in response to
surface stress, lacZ expression was induced with both
constructs (two to threefold over the corresponding basal
level) in wild-type but not in clgR deletion mutant cultures
(Fig. 2D). The presence of two promoters explains why
complementing the clgR deletion mutant restores only
∼ 40% of wild-type levels of the downstream genes
(Fig. 2B): In the complemented mutant, ClgR produced
from an ectopic locus can induce the stress response of
pspA-rv2743c-rv2742c only from the downstream promoter but not from the upstream promoter. To map the two
promoters, we analyzed data obtained by genome-wide
transcription start site (TSS) mapping using RNA adapter
ligation and next generation RNA sequencing (Shell et al.,
2015) (see Methods). A TSS was identified 30 nucleotides
upstream from the start of the clgR coding sequence
(Fig. 2E). The corresponding -10 and -35 sequences are
characteristic of a SigE promoter, which had been previously identified (Manganelli et al., 2001). An additional 5′
end associated with a strong SigA -10 motif was identified
51 nucleotides upstream of the pspA coding sequence
(Fig. 2E) [no -35 site was identified, consistent with the
reported absence of a consensus -35 site for M. tuberculosis SigA promoters (Feklistov and Darst, 2011; Cortes
et al., 2013)]. The two transcriptional start sites identified
here were also reported in a previous genome-wide
mapping study employing a different methodology (Cortes
et al., 2013). Overall, the results reported in this section
strongly suggest that one or more multicistronic transcripts are expressed from two stress-responsive, clgRdependent promoter sequences, one located upstream of
clgR and another found in the clgR-pspA intergenic
region. In agreement with the genetically assessed clgR
dependence of both promoters, assessed genetically,
ClgR-binding sites were found upstream of clgR (as also
reported in Estorninho et al., 2010) and of pspA (Fig. S2)
by bioinformatics.
Regulation of ClgR by downstream genes
Since ClgR is the regulator of the clgR-pspA-rv2743crv2742c region, we characterized its own regulation.
Induction of clgR by surface stress requires functional
sigE (Fig. S3 and Manganelli et al., 2001). To take into
account potential post-transcriptional regulatory events,
we assessed ClgR activity by using as read-out the
expression of the target gene clpP1 (rv2461c), which is
Fig. 3. Effect on clpP1 stress response of clgR deletion and
sequential complementation with clgR and downstream operon
genes. Expression of the sentinel target gene clpP1 was used as
readout of ClgR activity (Sherrid et al., 2010). Complementation of
the clgR deletion was performed sequentially by introducing clgR
alone, clgR plus pspA, plus pspA-rv2743c and plus
pspA-rv2743c-rv2742c. Transcript copy numbers were generated
and expressed as described in the legend to Fig. 2C. The reason
why clgR alone complements the clpP1 expression defect of the
clgR deletion mutant to similar levels as the
clgR-pspA-rv2743c-rv2742c complementation can be explained by
ectopically produced ClgR inducing pspA-rv2743c-rv2742c
expression from the promoter located in the clgR-pspA intergenic
region (Fig. 2D). Measurements of clpP1 transcripts performed
using M. tuberculosis H37Rv and M. tuberculosis CDC1551 gave
indistinguishable results (Fig. S8D), indicating that the clpP1
surface-stress response is conserved in different M. tuberculosis
strains.
directly induced by ClgR (Sherrid et al., 2010). Stress
(SDS)-mediated induction of clpP1 exhibited a biphasic
temporal expression profile, with one peak at ∼ 60–90 min
poststress and a second at ∼ 180–240 min. The two
peaks were separated by a trough at ∼ 120 min poststress
(Fig. 3). Induction of clpP1 was absent in a clgR deletion
mutant (Fig. 3). When we complemented the clgR mutant
with clgR alone (by using an ectopic, integrated copy of
the complementing DNA expressed from the native promoter), clpP1 induction was restored to wild-type levels at
most time points (Fig. 3). However, we were surprised to
find that mutant complementation with clgR and pspA
together abrogated clpP1 expression (Fig. 3): Addition of
pspA to the complementing DNA inhibited ClgR activity.
The expression profile of clpP1 was fully restored only
when both downstream genes, rv2743c and rv2742c,
were added to the clgR-pspA complementing DNA
(Fig. 3). Similar results were obtained when we measured
expression of another ClgR target gene, hsp (rv0251c)
(Estorninho et al., 2010) (Fig. S4A). Together, these
genetic data show that pspA has an inhibitory effect on
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 5
showed a striking decrease in the second peak of the
clpP1 profile (Fig. S5). Thus, pepD has a temporal effect
on ClgR activity similar to that previously described for the
rv2743c insertion mutant (Fig. 4A).
Protein–protein interactions
Fig. 4. Effects of rv2743c inactivation on target gene expression
and ATP levels.
A. Effect on clpP1 stress response. ClgR activity was assessed as
expression of the sentinel target gene clpP1 in an rv2743c
transposon (Tn) insertion mutant. Complementation was performed
with rv2743c-rv2742c to overcome the polar effect of the mutation
on the downstream rv2742c (data not shown). Wild-type, mutant
and rv2743c-rv2742c complemented (compl.) strains were
subjected to surface stress with SDS, and clpP1 transcripts were
enumerated and presented as described in the legend to Fig. 2C.
The reason why the rv2743c mutation did not phenocopy the
clgR-pspA complementation of the clgR deletion mutant (Fig. 3)
can be gene dosage effect due to the extra copy of pspA in the
complementing DNA.
B. Effect on intracellular ATP levels. ATP levels were assayed in
extracts from mid-log cultures of wild-type, mutant and
complemented strains pre- and post-treatment with 0.03% SDS for
24 h, and expressed as in the legend to Fig. 1A.
ClgR activity and that rv2743c and rv2742c reverse that
effect. Functional data were consistent with the genetic
results, as complementation of the clgR defect by the
clgR-pspA-rv2743c-rv2742c DNA also restored wild-type
levels of ATP in stressed cultures (Fig. S4B).
The effect of rv2743c-rv2742c on ClgR activity was
further examined with a mutant of rv2743c carrying a
transposon insertion that has a polar effect on the downstream gene rv2742c (data not shown). In this mutant, the
second peak of the clpP1 profile disappeared (Fig. 4A) (the
biphasic profile of the clpP1 response and the temporal
effects of the rv2743c-rv2742c mutation are interpreted in
the Discussion). In addition, the rv2743c-rv2742c mutation
reduced intracellular ATP levels under surface-stress conditions to < 60% of wild-type levels (Fig. 4B), thereby recapitulating functional effects observed with the clgR mutant
(Fig. 1).
Effect of the pepD protease gene on ClgR activity
In light of the inhibitory effect of pspA on ClgR activity,
described above with the clgR complementation studies
(Figs 3 and S4A), we reasoned that ClgR target gene
expression profiles might be affected by mutations in
accessory genes that alter PspA levels. One such gene is
pepD (rv0983), which encodes a serine protease that
targets PspA for proteolytic degradation (White et al.,
2011). Indeed, in a pepD-deficient mutant, the SDSmediated stress response of the ClgR target gene clpP1
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
We next determined whether protein complexes form
between ClgR, PspA and the Rv2743c and Rv2742c proteins. We constructed E. coli strains expressing IPTGinducible, differentially tagged copies of each protein in all
pairwise combinations, and then we assessed protein
complex formation by immunoprecipitation, pull-down
assays and western blot analyses. We detected complex
formation between ClgR and PspA (Fig. 5A, lane 3) but
Fig. 5. Protein–protein interactions. Pairwise protein–protein
interactions were assessed between differentially tagged
recombinant proteins expressed from IPTG-inducible promoters in
E. coli. Whole cell lysates from IPTG-induced recombinant cultures
expressing tagged proteins, alone or in pairs as indicated, were
used for co-immunoprecipitation (IP; left column) or for pull-down of
6xHis tagged protein (PD; right column), followed by
immunoblotting (IB) with the appropriate monoclonal antibody.
A–C. Left column: IP was performed with anti-His (A and C) or with
anti-S-tag antibody (B), followed by IB with anti-Myc antibody to
detect ClgR. Lanes: (1) whole-cell lysate + IB (positive control); (2)
IP (with cell extracts carrying ClgR-Myc alone) + IB; (3) IP (with cell
extracts carrying ClgR-Myc plus a His-tagged or S-tagged
protein) + IB.
D–F. Right column: PD was performed with Ni-NTA agarose,
followed by IB with anti S-tag antibody for detection of Rv2743c (D
and E) or Rv2742c (F). Lanes: (1) whole-cell lysate + IB (positive
control); (2) PD (with cell extracts carrying S-tag protein
alone) + IB; (3) PD (with cell extracts carrying S-tag protein plus
His-tag protein) + IB. The bottom row of each panel shows
unidentified E. coli bands cross-reacting with anti-Myc monoclonal
antibody (panels in the left column) or with the anti-S-tag
monoclonal antibody (panels in the right column) that served as
loading controls. Experiments in panels B, C and F were repeated
by switching tags in each corresponding protein pair to control for
potential tag effects on the protein–protein interaction, with no
change of result. H, His tag; M, Myc tag; S, S tag.
6 P. Datta et al. ■
observed on ClgR activity with the same mutant (Fig. 4A).
In addition, the rv2743c mutation reduced the mprA but
not the sigB stress response profile in the first 90 min
following SDS addition, suggesting that the effect is
directly on MprA activity and indirectly (through the dampening of MprA) on σE activity. Together, the observed
effects of the rv2743c mutation on surface stress tolerance and on the activity of the MprA-σE network establish
a link between the clgR-pspA-rv2743c-rv2742c region
and expression of envelope stress-sensing functions.
Fig. 6. Effects of rv2743c inactivation.
A. Tolerance to surface stress. Lethal activity of the detergent SDS
was determined in wild-type, rv2743c mutant and rv2743c-rv2742c
complemented (compl.) strains. Data are expressed as % survival
relative to untreated cultures. Mean values (± standard error of the
mean) from triplicate experiments are shown.
B. Surface-stress response of sentinel target genes of MprAB and
σE. Transcripts for mprA and sigB were enumerated in wild-type,
mutant and complemented strains and presented as described in
the legend to Fig. 2C. Tn, transposon.
not between ClgR and either Rv2743c (Fig. 5B, lane 3) or
Rv2742c (Fig. 5C, lane 3). Moreover, protein complexes
formed between PspA and Rv2743c (Fig. 5D, lane 3) and
between Rv2743c and Rv2742c (Fig. 5E, lane 3), but not
between PspA and Rv2742c (Fig. 5F, lane 3). In summary,
protein complexes formed only between ClgR and PspA,
PspA and Rv2743c, and Rv2743c and Rv2742c.
Preservation of envelope integrity
Since clgR and rv2743c mutants show reduction of cellular processes that are typically associated with envelope
integrity, such as energy generation under stress (Figs 4B
and S4B), we asked whether the clgR-pspA-rv2743crv2742c region has an envelope-preserving activity. Since
envelope integrity is expected to be linked to surfacestress tolerance, we measured survival of M. tuberculosis
wild-type and rv2743c mutant cultures during treatment
with bactericidal concentrations (0.2%) of SDS. Mutant
survival was drastically lower (> 90% reduction) than wildtype survival (Fig. 6A). A similar effect was observed with
a clgR mutant (data not shown).
Another measure of envelope integrity is proper functioning of the envelope stress-sensing system, which, as
pointed above, centers on the MprAB-σE network. We
examined the effects of genetic inactivation of rv2743c on
the MprAB-σE network. As read-out for MprAB and σE
activity, we measured expression of mprA [which contains
regulatory MprA-binding sites (He et al., 2006)] and sigB
[which is transcribed from a σE-dependent promoter
(Song et al., 2008)]. Inactivation of rv2743c reduced mprA
and sigB expression to ∼ 20% and ∼ 10% of wild-type
levels, respectively, after 90–120 min of treatment with
SDS (Fig. 6B). This temporal effect was similar to that
Mycobacterium tuberculosis survival in macrophages
Given that the ClgR-PspA-Rv2743c-Rv2742c module
maintains tolerance to surface stress in vitro, we tested
whether the intact system is required for M. tuberculosis
survival during macrophage infection. When rv2743c was
inactivated, M. tuberculosis growth in primary human
monocyte-derived macrophages (MDMs) was reduced
40% at 7 days postinfection (Fig. 7A). Since mutant and
wild-type cultures are indistinguishable when grown in
rich medium, the rv2743c inactivation increases M. tuberculosis susceptibility to the intramacrophage environment. Similar effects were observed with a clgR mutant
(Fig. 7B). Thus, the clgR-pspA-rv2743c-rv2742c region
facilitates intracellular growth of M. tuberculosis.
Comparison with the Psp proteins of
Gram-negative bacteria
We next asked whether the proteins encoded by the clgRpspA-rv2743c-rv2742c operon of M. tuberculosis share
similarities with the proteins mediating the Psp response
Fig. 7. Effect of rv2743c and clgR inactivation for growth in
macrophages. Human monocyte-derived macrophages were
infected with different M. tuberculosis strains. Bacterial counts (cfu)
were normalized to 105 adherent cells per well. Data are expressed
as fold increase in cfu at day 7 relative to cfu enumerated at 4 h
postinfection. Mean values (± standard error of the mean) from
triplicate experiments are shown.
A. Wild-type, rv2743c mutant and rv2743c-rv2742c complemented
(compl.) strains. Tn, transposon.
B. Wild-type, clgR mutant and complemented (compl.) strains.
Complementation was with clgR-pspA-rv2743c-rv2742c DNA.
Reduced growth of the clgR mutant in murine bone marrow-derived
macrophages was previously reported (Estorninho et al., 2010).
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 7
Fig. 8. Mycobacterium tuberculosis (Mtb) and E. coli (Eco) Psp protein comparison.
A. Mtb ClgR and Eco PspF. ClgR and PspF contain helix-turn-helix (HTH)-type DNA-binding domains (DBDs). ClgR has an N-terminal
HTH_XRE (SM00530, PF01381) DBD, while PspF has a C-terminal Fis-type HTH_8 DBD (PF02954, IPR002197). PspF contains AAA+ and
sigma-54 interaction domains (IPR003593 and IPR002078 respectively).
B. Mtb PspA and Eco PspA. Both proteins were used to define PspA/IM30 (PF04012, IPR007157), a domain characterized by ∼ 25% of
conserved amino acid residues spanning the entire protein length. This domain is also predicted to contain coiled-coils.
C. Mtb Rv2743c and Eco PspB. Mtb Rv2743c and Eco PspB do not share significant sequence similarity; however, they are both predicted to
be transmembrane (TM) proteins. Mtb Rv2743c is predicted to have two ∼ 22-amino-acid TM helices connected by a short (4–6 amino acid)
noncytoplasmic loop. Mtb Rv2743c is also predicted to contain a C-terminal coiled-coil. Eco PspB is a single PspB domain (PF06667,
IPR009554), which contains a predicted N-terminal TM helix (residues: 6–24) and C-terminal coiled-coil (residues: 36–64).
D. Mtb Rv2742c and Eco PspC. Mtb Rv2742c and Eco PspC share no significant sequence similarity. Sequence analysis of Rv2742c
revealed only a single domain of unknown function (DUF3046, PF11248), which is conserved in Actinobacteria. In contrast, Eco PspC has an
N-terminal PspC domain (residues: 7–68, PF04024), a transmembrane helix (residues: 39–64) and a C-terminal coiled-coil (residues: 84–105).
to envelope stress in Gram-negative Proteobacteria
(reviewed in Darwin, 2005; Joly et al., 2010; Darwin, 2013).
In that system, the ‘minimal’ Psp module (Darwin, 2005)
includes four adjacent genes, namely, the regulator pspF
and the PspF-regulated pspABC. Thus, the gene organization in the Gram-negative module parallels that seen in
M. tuberculosis. Except for PspA, which contains the
PspA/IM30 domain characteristic of this protein family
(Fig. 8), the other proteins encoded by the clgR-pspArv2743c-rv2742c region share no sequence homology with
the Psp proteins of Gram-negative bacteria (Fig. 8). The
transcriptional regulators ClgR and PspF are very different
in length. Moreover, ClgR lacks the AAA+ ATPase domain
characteristic of bacterial enhancer binding proteins such
as PspF, and the respective helix-turn-helix DNA binding
domains are located at opposite ends of the two proteins
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
(Fig. 8). In addition, while PspB and PspC are both integral
membrane proteins, only Rv2743c (but not Rv2742c) contains transmembrane domains (Fig. 8). Thus, the proteins
encoded by clgR, rv2743c and rv2742c of M. tuberculosis
are unrelated to the Psp system of Gram-negative organisms with respect to amino acid sequence.
Phylogenetic analysis
The distribution of the Psp system and its components in
species encoding ClgR homologs was not investigated in
previous phylogenetic analyses of the Psp system (e.g.
Joly et al., 2010), presumably due to the absence of
sequence homology between the regulators PspF and
ClgR (Fig. 8). We performed protein sequence similarity
searches in ∼ 3400 species of Actinobacteria and found
8 P. Datta et al. ■
Fig. 9. Distribution of
ClgR-PspA-Rv2743c-Rv2742c proteins in
Mycobacterium spp. Significant matches and
similarity scores were obtained as described
in Methods. The heat map represents the
similarity score per target species or
sub-species (or the highest score in the case
of multiple matches), expressed as percent of
protein sequence similarity for the longest
contiguous match. Mycobacterial species
were ordered on the basis of phylogenetic
similarity (Wattam et al., 2014 and
http://www.patricbrc.org). The box contains
the species in the M. tuberculosis complex.
Intergenic distances (IG) between matches
were calculated (in bp) in the target genomes.
IG1, IG2 and IG3 denote distances between
clgR-pspA, pspA-rv2743c and
rv2743c-rv2742c respectively. Filled black
circles indicate intergenic distances that are
similar to that of M. tuberculosis
clgR-pspA-rv2743c-rv2742c operon, as shown
in Fig. 2A. Open circles denote either
absence of one gene in the pair, intergenic
distances of > 250 bp or absence of genomic
location information. *Of 13 virulent M. bovis
isolates analyzed, one (AF2122/97) contained
a 23-bp insertion in pspC approximately
two-thirds into the gene sequence,
presumably giving rise to a truncated product.
The same insertion was found in the
genomes of nine out of 14 substrains of the
attenuated vaccine strain M. bovis Bacillus
Calmette Guerin. The evolutionary
significance of this polymorphism remains to
be ascertained. **Only Mycobacterium leprae
was devoid of the entire ClgR-Psp module, as
previously reported (Bretl et al., 2014),
suggesting that loss of the Psp system is part
of the genomic downsizing observed with this
obligate intracellular pathogen (Cole et al.,
2001). ***Only Mycobacterium avium among
nontuberculous mycobacteria showed a
truncated form of Rv2742c (∼ 1/3 the length of
M. tuberculosis Rv2742c) at the appropriate
distance from the preceding gene.
that ∼ 2200 species carried a protein having at least 25%
homology with M. tuberculosis ClgR (E-value < 1e-5).
When we examined the ClgR-positive species for conservation of PspA-Rv2743c-Rv2742c, we found that the
number of genes present correlated inversely with phylogenetic distance from Mycobacterium spp. (Fig. S6).
Within Mycobacterium spp., only the M. tuberculosis
complex contains the entire ClgR-PspA-Rv2743cRv2742c region: Mycobacteria outside the M. tuberculosis
complex showed < 85% homology with Rv2743c and
lacked Rv2742c (Fig. 9). Despite the lack of a fourth,
contiguous gene in the module, the nontuberculous
species Mycobacterium smegmatis showed induction of
clgR and the ClgR target clpP1 in response to surface
stress (Fig. S7), and expression profiles resembled those
seen in M. tuberculosis (Figs 2C and 3). Thus, nontuberculous mycobacteria encode three contiguous genes that
appear to be sufficient for regulation of the module’s genes
(we cannot exclude other, noncontiguous gene(s) participating to the Psp function). Overall, our analysis shows that
the four-gene module is characteristic only of tuberculous
mycobacteria.
Discussion
The present work connects envelope stress signaling with
envelope maintenance in M. tuberculosis, thus completing
the circle between sensing stress and recovering from
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 9
damage. Our finding that the four-gene clgR-pspArv2743c-rv2742c module is conserved only within the
M. tuberculosis complex strongly suggests a link between
this module and the ability of tuberculous mycobacteria to
cause disease. Such linkage is consistent with the reduced
intramacrophage growth of M. tuberculosis we find associated with disruption of the clgR-pspA-rv2743c-rv2742c
operon.
We found strong similarities between the clgR-pspArv2743c-rv2742c operon of M. tuberculosis and the Psp
system of Gram-negative bacteria. For example, the
minimal Psp module includes four adjacent genes, the
regulator pspF and its three regulated genes, pspABC. In
M. tuberculosis, the regulator is clgR, followed by pspArv2743c-rv2742c. With the Gram-negative system, the
regulator’s transcriptional activity is normally blocked by
formation of the PspF-PspA complex; envelope stress
presumably alters PspA conformation and leads to formation of higher-order PspA multimers (Joly et al., 2010).
The resulting multimeric structure results in (i) release of
PspF, (ii) consequent PspF-dependent induction of the
pspABC operon by σ54-containing RNA polymerase, and
(iii) formation of a complex between PspA and the two
integral membrane proteins PspB and PspC (Darwin,
2005; Joly et al., 2010). With M. tuberculosis, our data fit
with a model (Fig. 10A) that parallels the Gram-negative
scenario. We propose that surface-stress-dependent
induction of the clgR-pspA-rv2743c-rv2742c operon by
MprAB-σE results in formation of a complex between ClgR
and PspA. This complex would block ClgR activity at
∼ 90 min post-stress (Fig. 3). A second peak of ClgR activity (∼ 120 min onwards) requires functional rv2743crv2742c (Fig. 4A), which we interpret as PspA forming a
multiprotein complex with Rv2743c-Rv2742c and releasing ClgR from the inhibitory complex (this proposition
implies that the pair-wise interactions observed with
recombinant proteins (Fig. 5) reflect the ability to form a
multiprotein complex in living cells). Thus, the ubiquitous
protein PspA would exert parallel inhibitory function over
the cognate regulator in M. tuberculosis (ClgR) and in
Gram-negative organisms (PspF). Moreover, since PspA
repurposing from inhibitor to effector activity in Gramnegative bacteria requires PspBC, these two proteins
positively regulate PspF activity (Joly et al., 2010). Likewise, the M. tuberculosis rv2743c-rv2742c genes positively affect ClgR activity (Fig. 4A). Thus, despite the
absence of amino acid sequence homology, Rv2743c and
Rv2742c might serve as functional homologs of PspB and
PspC of Gram-negative organisms. Indeed, the disappearance of the second peak of ClgR activity in a mutant
deficient in pepD (Fig. S5), which normally targets PspA
for degradation (White et al., 2011), supports our model:
wild-type abundance of Rv2743c-Rv2742c may be insufficient to repurpose excess PspA found in the pepD
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
mutant, leading to continuing inhibition of ClgR activity by
PspA. These regulatory similarities between phylogenetically distant microorganisms emphasize the widespread
importance of the four-gene Psp module.
Parallels also exist between the Gram-negative and
the M. tuberculosis systems in terms of both function and
sub-cellular localization. The membrane-bound PspABC
complex of Gram-negative bacteria has envelopestabilizing activities, with PspA exhibiting key effector
functions (Darwin, 2005; Darwin, 2013; Joly et al., 2010).
Similar effector functions are presumably exerted by the
M. tuberculosis module since clgR-pspA-rv2743crv2742c is induced when PMF is dissipated and is
required to express phenotypes diagnostic of envelope
integrity, such as ATP homeostasis and tolerance to
surface stress. We note that the extensive network of
positive feedback loops regulating this system (Fig. 10B)
prevents genetic analyses from formally assigning these
functional phenotypes to direct (envelope-preserving)
effects or to indirect (ClgR activity modulating) effects of
the pspA-rv2743c-rv2742c genes. As with Gramnegative bacteria, the multiprotein complex between
PspA, Rv2743c and Rv2742c may be targeted to the
M. tuberculosis cell envelope since Rv2743c is a membrane protein (Fig. 8). Intriguingly, sub-cellular fractionation analyses of M. tuberculosis have revealed both
PspA- and Rv2742c-derived peptides in the cell membrane fraction, despite the absence of transmembrane
domains or lipid anchoring motifs in these proteins
(Rumschlag et al., 1990; Mawuenyega et al., 2005).
These observations support the presence in envelopestressed mycobacterial cells of an envelope-bound, multiprotein complex containing PspA, Rv2743c and
Rv2742c proteins, parallel to the Gram-negative situation. The observation that clgR and its target genes
are induced in response to surface stress in M. smegmatis (Fig. S7), which encodes only three of the four
genes found in the Psp module of the M. tuberculosis
complex (Fig. 9), provides an opportunity for parallel
functional and structural analyses of the Psp module
between tuberculous and nontuberculous mycobacteria,
and between mycobacteria and Gram-negative bacteria.
In conclusion, our work reveals parallel or convergent
evolution of the Psp response in bacteria of different
phyla in which conserved functions are expressed by
unrelated proteins. The Gram-negative and M. tuberculosis systems have, however, evolved dynamic differences.
In Gram-negative bacteria, pspF is constitutively
expressed (Weiner et al., 1991), and the PspF-PspA
complex is found in unstressed cells (Darwin, 2005; Joly
et al., 2010). In M. tuberculosis, clgR is stress-induced,
and our data point to formation of the ClgR-PspA complex
in response to stress. The simpler, prestress/poststress
dichotomy seen in Gram-negative bacteria may allow a
10 P. Datta et al. ■
Fig. 10. A. Model of MprAB-σE-dependent maintenance of envelope integrity in response to surface stress. The figure shows interactions
between the envelope-stress signaling MprAB-σE network and the envelope-preserving ClgR-PspA-Rv2743c-Rv2742c system. Interactions
include the mprAB-sigE transcriptional network and the mprAB-dependent pepD, the downstream target clgR-pspA-rv2743c-rv2742c operon
and additional clgR-regulated genes (clpP1). The line plot represents the temporal, poststress expression profile of clpP1, as a proxy of ClgR
activity. The location of individual proteins, protein complexes and interactions relative to the line plot depicts the proposed temporal course of
events associated with propagation of signal (ClgR activity) through the Psp system, resulting membrane integrity and MprAB stabilization.
The scissors icon indicates proteolytic degradation. The red cross over the ClgR activity icon represents ClgR inactivation.
B. Feedback loops in the stress-response system of M. tuberculosis. The wiring diagram shows multiple regulatory feedback loops,
transcriptional and post-transcriptional, operating between mprAB, sigE, clgR, the Psp system, and neighboring network genes and/or
corresponding gene products under surface stress conditions. The abundance of feedback loops in the system does not allow distinguishing
whether the observed functional effects of the pspA-rv2743c-rv2742c products are direct or occur indirectly via the regulation of ClgR and its
target gene products. Since clgR is connected to the mprAB/sigE sensing network through two positive feedbacks, links between
envelope-stress-sensing and envelope-preserving functions exist regardless of whether pspA-rv2743c-rv2742c act directly or indirectly. Except
for sigE autoregulation (Chauhan and Gennaro, unpublished), the regulatory interactions showed in this figure are all cited or discovered in the
present manuscript. Arrowhead = positive regulation; Barhead = negative regulation.
faster response. In contrast, poststress formation of the
inhibitory ClgR-PspA complex in M. tuberculosis, together
with the positive feedback loops connecting the ClgRPspA-Rv2743c-Rv2742c system with the MprAB-σE
network (Fig. 6B), may be better suited for sustained
responses to multiple, envelope-perturbing signals that are
likely to occur over the long course of chronic infection.
Such dissimilar dynamics point to how conserved functions
can adapt to different bacterial life styles by varying regulatory modes.
Experimental procedures
Bacterial strains, reagents and growth conditions
Escherichia coli XL1 blue (Agilent Technologies, Santa Clara,
CA, USA) was used for DNA cloning. E. coli BL21(DE3)
(Novagen, La Jolla, CA, USA) was used for recombinant
protein expression. All E. coli strains were grown with aeration
at 37°C in Luria-Bertani (LB) broth or agar (Thermo Fisher
Scientific, Waltham, MA, USA). Media were supplemented
with 25 μg ml−1 chloramphenicol (Sigma, St. Louis, MO, USA),
150 μg ml−1 hygromycin B (Roche Diagnostic, Mannheim,
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 11
Germany), 100 μg ml−1 ampicillin and 50 μg ml−1 kanamycin
sulfate (Thermo Fisher Scientific), as needed. M. tuberculosis
knock-out mutants in sigE, clgR and pepD and corresponding
complemented strains were previously reported (Manganelli
et al., 2001; Estorninho et al., 2010; White et al., 2010). A
transposon-insertion mutant in gene rv2743c was obtained
from the BEI repository (NR18017; http://www.beiresources
.org/Catalog). Complemented strains for clgR and rv2743c
mutants were constructed in this work (described next in
DNA manipulations). Gene numbering for M. tuberculosis is
according to Cole et al. (1998). M. tuberculosis cultures were
grown in Dubos Tween-albumin broth (Becton Dickinson,
Sparks, MD, USA) (liquid medium) or 7H10 (solid medium)
(Difco, Franklin Lakes, NJ, USA) supplemented with 0.05%
Tween 80, 0.2% glycerol and 10% ADN (2% glucose, 5%
bovine serum albumin, 0.15 M NaCl). Liquid cultures of
M. tuberculosis were grown in 25 ml tubes at 37°C with
magnetic-bar stirring at 450 r.p.m. Plates were incubated at
37°C in sealed plastic bags. Both solid and liquid media were
supplemented with 25 μg ml−1 kanamycin sulfate (ThermoFisher Scientific, Waltham, MA), 50 μg ml−1 hygromycin B,
as needed. M. smegmatis cultures were grown in 7H9 broth
(Difco) supplemented with 0.05% Tween 80 and 0.2%
glycerol.
DNA manipulations
Construction of complementing plasmids: A clgR knock-out
mutant was complemented with DNA comprising an increasing number of open reading frames. These fragments were
obtained by using the same forward primer (mapping 382 bp
upstream of the clgR coding sequence) paired with a different
reverse primer mapping at the 3′end of clgR, pspA, rv2743c
or rv2742c to amplify the DNA sequences encompassing
clgR, clgR-pspA, clgR-pspA-rv2743c or clgR-pspA-rv2743crv2742c respectively. Amplified fragments were cloned in an
integrative E. coli-mycobacteria shuttle vector pMV306kan
(Stover et al., 1991; Braunstein et al., 2001). To take into
account the polar effect of the rv2743c mutation on rv2742c,
a DNA fragment encompassing clgR-pspA-rv2743c-rv2742c
was generated for complementation of the rv2743c mutant
with rv2743c-rv2742c expressed from the native promoters,
and cloned in the integrative plasmid pMV306Hyg. To inactivate clgR and pspA in this complementing DNA, an in-frame
deletion was introduced in pspA, and an in-frame stop codon
was introduced in clgR, in the following way. Genomic DNA
was used as template to amplify two DNA fragments: (i) a
895 bp fragment spanning from 382 bp upstream of clgR to
45 bp downstream of pspA and (ii) a 1848 bp fragment spanning from 771 bp downstream of pspA to 69bp downstream
of the rv2742c ORF. The first fragment was further used as
template for site-directed mutagenesis [performed by overlap
extension PCR (Datta et al., 2002)] to introduce an in-frame
stop codon (TAA) 54 bp downstream of the start codon of the
clgR open reading frame. The fragment containing the
in-frame mutation of clgR was cloned at the KpnI-XbaI sites,
while the second fragment was cloned at the XbaI-HindIII
sites in plasmid pMV306-Hyg. Cloning these two fragments
next to each other resulted in a 723 bp in-frame deletion in
pspA. Thus, the resulting fragment contained the entire clgRpspA-rv2743c-rv2742c region in which both clgR and pspA
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
were nonfunctional. For complementation of the hsp mutant,
a DNA fragment spanning the hsp coding sequence and the
upstream 400 bp region was amplified by PCR. The amplified
fragments were cloned in the integrative plasmid pMV306Hyg. In all cases, the nucleotide sequence of the complementing DNA fragment was verified by DNA sequence
analysis. Nucleotide sequences of PCR primers are listed in
Table S1. Constructs were introduced in the corresponding
mutant strains by electroporation. Transformants were
selected on plates supplemented with appropriate antibiotics
and verified by PCR analysis.
Construction of lacZ fusions: For construction of
promoter::lacZ fusions, DNA fragments containing sequences
upstream of clgR (382 bp) and upstream of pspA (250 bp) plus
the first eight codons of the corresponding open reading frame
were amplified. Amplified fragments were cloned in frame with
lacZ in the promoter probe E. coli-mycobacteria shuttle
plasmid pJEM13 (Timm et al., 1994). Nucleotide sequences of
PCR primers are listed in Table S1. The resulting plasmids
were introduced by electroporation in M. tuberculosis wildtype and clgR mutant. Transformants were selected on 7H10
agar plates supplemented with kanamycin.
SDS treatment
For gene expression analyses, mid-log cultures of M. tuberculosis were treated with SDS to select a bacteriostatic concentration of the detergent (0.03%) (Fig. S8A) that induced
sigE (Fig. S8B), and had no lethal effect in a 6 h treatment on
any of the mutants used (Fig. S8C). Following treatment with
0.03% SDS, 1 ml culture aliquots were collected at various
time intervals up to 4 h and used for RNA extraction. For
assessment of relative susceptibility to bactericidal SDS concentrations, wild-type and mutant M. tuberculosis cultures
were grown to mid-log phase, and aliquots of approximately
105 cells were treated for 24 h with a bactericidal concentration of the detergent (0.2%) (Fig. 6A). After incubation,
samples were diluted and plated on stressor-free medium to
determine the fraction of surviving cells relative to samples
taken at the time of SDS treatment.
CCCP treatment
Mid-log-phase cultures of M. tuberculosis were treated with
CCCP at increasing concentrations (up to 100 μM). Since
CCCP is dissolved in DMSO, a solvent-only control (corresponding to the highest DMSO concentration used with the
CCCP treatment) was also included. Following 6 h of treatment, 1 ml culture aliquots were collected and used for RNA
extraction.
RNA extraction and enumeration of bacterial transcripts
Bacterial cell pellets were resuspended in 1 ml TRI reagent
(Molecular Research Center, Cincinnati, OH, USA) and 0.8 ml
zirconia beads (0.1 mm diameter, BioSpec Products, Inc.,
Bartlesville, OK, USA). Cells were disrupted in a bead-beater
(Mini-Beadbeater-16, BioSpec Products) by three 45 s pulses,
each separated by 10 min incubation on ice. Cells were lysed
by adding 100 μl BCP Reagent (Molecular Research Center)
and vigorous mixing for 10 minutes. After 5 min incubation at
12 P. Datta et al. ■
room temperature, the tubes were centrifuged for 30 min at
12 000 × g at 4°C. The aqueous phase was transferred to
fresh tubes containing 500 μl isopropanol for overnight precipitation. After four cycles of overnight precipitation, samples
were washed with 75% ethanol, air-dried and resuspended in
diethyl pyrocarbonate-treated H2O for storage at −80°C.
Reverse transcription was performed with random hexameric
primers and ThermoScript Reverse Transcriptase (Invitrogen,
Carlsbad, CA, USA). Enumeration of mRNAs was carried out
by qPCR using gene-specific primers, molecular beacons and
AmpliTaq Gold polymerase (Applied Biosystems, Foster City,
CA, USA) in a Stratagene Mx4000 thermal cycler (Agilent
Technologies, La Jolla, CA, USA).
Nucleotide sequences of PCR primers and molecular
beacons are listed in Table S1. The M. tuberculosis 16s rRNA
copy number was used as a normalization factor to enumerate bacterial transcripts per cell, as previously described (Shi
et al., 2003).
Escherichia coli constructs for protein expression
The coding regions for clgR, pspA, rv2743c and rv2742c of
M. tuberculosis were each amplified using gene-specific
primers (Table S1) and cloned into either of the compatible
plasmids pETDUET and pACYCDUET (EMD Chemicals, San
Diego, CA, USA) under an IPTG-inducible T7 promoter. A
fragment carrying the clgR coding sequence was cloned at the
MCS1 site of plasmid pACYCDUET to express a recombinant
product carrying a C-terminal Myc tag. The pspA coding
sequence was cloned at the MCS1 of pETDUET to obtain a
N-terminally 6xHis-tagged protein. The rv2743c coding
sequence was cloned at the MCS2 site in pETDUET or in the
pspA-pETDUET to obtain a C-terminally S-tagged protein.
The rv2742c coding sequence was cloned at the MCS1 site
of either pETDUET or rv2743c-pETDUET to obtain an
N-terminally 6xHis-tagged protein, and it was also cloned
at the MCS2 of pspA-pETDUET to be expressed as a
C-terminally S-tagged protein. The resulting DNA was transformed into E. coli XL1 Blue. Transformants were selected on
antibiotic-containing LB agar plates and analyzed by restriction endonuclease mapping and DNA sequencing. For
expression of recombinant proteins, plasmids expressing differentially tagged protein pairs or each protein alone were
electroporated into E. coli BL21(DE3) cells. Transformants
expressing protein or proteins pair were selected on plates
supplemented with the appropriate antibiotics.
sequencing libraries were made from each RNA preparation: a
‘converted’ library, which captured RNA 5′ ends endogenously
bearing 5′ triphosphates or 5′ monophosphates, and a ‘nonconverted’ library, which captured only RNA 5′ ends endogenously bearing 5′ monophosphates. For the ‘converted’
library, RNA was pretreated with polyphosphatase (Epicentre,
Madison, WI, USA). Samples were purified by RNeasy
(Qiagen) and ligated to an adapter oligo SSS392 (TCCCTA
CACGACGCTCTTCCGAUCU; normal font indicates deoxyribonucleotides and italics indicate ribonucleotides) with T4
RNA ligase I (New England Biolabs, Ipswitch, MA, USA).
Ligation products were purified and sheared in a Covaris
sonicator. cDNA was synthesized with oligo SSS397 (CTG
GAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN, where
‘N’ represents a degenerate base) using Superscript III (Life
Technologies). Purified cDNA was subsequently amplified
by eight PCR cycles using primers containing additional
Illumina adapter sequences (oligo SSS398, AATGATAC
GGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC
TCTTC and a reverse oligo CAAGCAGAAGACGGCATACGA
GATXXXXXXGTGACTGGAGTTCAGACGTGTGCT, where
‘XXXXXX’ is a 6-nucleotide Illumina index sequence). PCR
products were size-selected by agarose gel electrophoresis to
a range of 150–500 nucleotides, purified with a Qiagen gel
extraction kit followed by Ampure XP beads (Beckman Coulter,
Pasadena, CA) and subjected to an additional four PCR cycles
with oligo SSS401 (AATGATACGGCGACCACCGAGATC)
and oligo SSS402 (CAAGCAGAAGACGGCATACGAGAT).
PCR products were purified with Ampure XP beads and
sequenced on an Illumina Genome Analyzer. 56-nucleotide,
paired-end reads were mapped using the sequence alignment
algorithm Ssaha2. Peaks in 5′-end coverage were identified
and filtered based on the absolute read count values in the
converted libraries as well as the ratio of these read counts to
coverage in RNAseq expression libraries. For each filtered
peak, the ratio of ‘1st nucleotide’ coverage in the converted/
nonconverted libraries was determined after pooling coverage
from replicate libraries. Gaussian mixture modeling of
bimodally distributed ratios was used to estimate means and
standard deviations for two skewed normal distributions (processed and unprocessed 5′ ends). RNA 5′ ends with ratios
> 1.74 had a cumulative probability of ≤ 0.01 of belonging to
the processed 5′ end population after Benjamini-Hochberg
adjustment and were designated TSSs.
Recombinant protein expression and
immunoprecipitation
TSS mapping
Replicate cultures of M. tuberculosis H37Rv were grown to
late-log phase (optical density ∼ 1) in 7H9 Middlebrook broth
supplemented with 10% OADC, 0.05% Tween-80 and 0.2%
glycerol. Cell pellets were disrupted in Trizol (Life Technologies, Carlsbad, CA, USA) in Lysing Matrix B tubes in a
FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA,
USA), and RNA was extracted according to the manufacturer’s
instructions. RNA was subsequently treated with DNase Turbo
(Life Technologies) and purified by RNeasy (Qiagen, Hilden,
Germany) before depletion of ribosomal RNA with a MICROBExpress kit (Life Technologies). TSSs were mapped as
described (Shell et al., 2015). Briefly, two parallel Illumina
To test protein–protein interactions, mid-log E. coli BL21(DE3)
cultures expressing two differentially tagged proteins singly or
together were treated with 200 μM IPTG for 2 h; 50 ml cultures
were harvested and subjected to lysis in 1 ml B-PER reagent
as per manufacturer’s protocols (Thermo Scientific, Rockford,
IL, USA). The whole cell lysate was clarified by centrifugation.
Total protein content of the cleared lysates was measured with
the Bradford assay. Cleared lysates containing fixed amounts
of total protein were incubated with monoclonal anti-His or anti
S-tag antibody following manufacturer’s protocols (EMD
Chemicals). Immune complexes were pulled down with 20 μl
protein A/G agarose (EMD Millipore, Billerca, MA, USA).
Agarose-bound complexes were washed twice with 200 μl
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
The Psp response of Mycobacterium tuberculosis 13
B-PER reagent, and the resulting samples were boiled in 20 μl
of 2× Laemmli sample buffer for 15 min. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes for
Western blot analysis with anti-Myc antibody (Life technology,
Grand Island, NY, USA).
Protein–protein interactions were also analyzed by pulldown assays. Cell lysates expressing recombinant 6xHis
tagged protein, alone or together with the potential interacting
partner, were incubated with Ni-NTA agarose (EMD Chemicals). Slurry was washed twice with 500 μl wash buffer (EMD
Chemicals), boiled with 2× Laemmli buffer and subjected to
SDS-PAGE. Western blot analysis was performed with anti-S
tag antibody, as above. Detection was carried out by incubation with anti-mouse IgG-HRP-conjugate (EMD Chemicals)
and enhanced chemiluminescence (Cell Signaling Technology, Danvers, MA, USA).
Measurement of ATP
Exponentially growing cultures were treated with 0.03% SDS
for 24 h. One milliliter aliquots of untreated and SDS-treated
cultures were harvested for total ATP measurement, as
described (Wayne and Hayes, 1996). Briefly, cell pellets were
resuspended in 0.025 M HEPES, pH 7.75 (supplemented
with 0.02% Tween-80). Aliquots of 0.05 ml of cell suspensions were diluted in equal volume of 0.025 M HEPES, and
0.04 ml of chloroform was added to the each tube. The resulting sample was heated at 80°C for 20 min, followed by addition of 4.9 ml of 0.025 M HEPES. A 0.05 ml aliquot of the
sample was mixed with an equal volume of the luciferinluciferase reagent, and light unit was recorded in a luminometer (Glomax-R, Promega Corporation, Madison, WI, USA).
Light units were converted to concentrations of ATP by use of
a standard curve of light units measured with various freshly
prepared dilutions of a 100 ng ml−1 ATP standard solution.
Concentration of ATP was normalized to total protein content.
Culture and infection of human MDMs
Human buffy coats were obtained from the New York Blood
Center (Long Island City, NY, USA), and peripheral blood
mononuclear cells (PBMCs) were prepared by Ficoll density
gradient centrifugation (Ficoll-Paque™, GE Healthcare,
Uppsala, Sweden) as described (Davies and Gordon, 2005).
Isolated PBMC were washed and resuspended in RPMI-1640
medium (Corning, Manassas, VA, USA) supplemented with
10% fetal bovine serum (Seradigm, Radnor, PA, USA) and
4 mM L-glutamine (Corning), plated at a density of 5 × 106
cells per well in 24-well plates and incubated overnight in
humidified atmospheric air containing 5% CO2 at 37°C to let
monocytes adhere, following standard protocols (e.g. Sharma
et al., 2009). Nonadherent cells were removed by washing
three times with 1× PBS; adherent cells were maintained in
supplemented RPMI-1640 medium and allowed to differentiate for 4 days in a 5% CO2 incubator at 37°C. At day 5, medium
was removed, and MDMs were counted and infected with
M. tuberculosis strains. Bacterial inoculum for infection was
prepared by diluting a frozen bacterial stock in supplemented
RPMI-1640 medium to obtain an MOI of 0.1 [colony forming
units (cfu) per cell]. Bacterial clumps were disrupted by vortexing with sterile 3 mm diameter glass beads for 2 min, and
this suspension was used for MDM infection. After 4 h of
© 2015 John Wiley & Sons Ltd, Molecular Microbiology
infection, extracellular bacteria were removed by washing
three times with 1× PBS, and fresh supplemented RPMI-1640
medium was added to cells. Medium was replenished also at
days 2 and 5 postinfection. At 4 h (to determine infecting dose)
and at day 7 postinfection, bacterial CFU were enumerated by
lysing infected MDM with 0.05% SDS and plating serial dilutions of cell lysates on agar plates. Prior to lysis, an aliquot of
cells was used to calculate the number of adherent cells in
each well, and CFU were normalized to 105 adherent cells.
In silico nucleotide and amino acid sequence analyses
To identify putative ClgR binding sites, ClgR binding
sequences were obtained from (Estorninho et al., 2010).
MEME software (Bailey and Elkan, 1994) was used to identify
all binding sites and to generate the corresponding motifs and
the sequence logo. The tool MAST (Bailey and Gribskov,
1998) was used to calculate the P values associated with the
sequence matches.
Analysis of primary amino acid sequence similarity was
performed using the Blosum62 matrix in Geneious Pro (Biomatters Ltd., Auckland, New Zealand). Comparative modeling
and consensus secondary structure predictions were carried
out using Phyre (Kelley and Sternberg, 2009), which incorporates the results of calculations made using Psi-Pred
(McGuffin et al., 2000), SSPro (Pollastri et al., 2002) and
Jpred (Cole et al., 2008). Additional protein and domain classification was carried out using InterPro (Hunter et al., 2012),
InterProScan (Jones et al., 2014), Pfam (Sonnhammer et al.,
1997; Mistry et al., 2007; Finn et al., 2011) and SMART
(Schultz et al., 1998; Letunic et al., 2014). Coiled-coil predictions were done using Coils (Lupas et al., 1991). Transmembrane regions were predicted using Phobius (Kall et al., 2004),
TMHMM (Sonnhammer et al., 1998), HMMTop (Tusnady and
Simon, 1998; 2001) and TMPred (Hofmann and Stoffel, 1993).
For the phylogenetic analyses, protein sequence similarity
searches were carried out using NCBI BLAST (Altschul et al.,
1990; 1997) with each of M. tuberculosis clgR-pspABC proteins as query against a set of 3436 genomic sequences from
Actinobacteria (taxid: 201174) obtained from the PATRIC
database (Wattam et al., 2014 and http://www.patricbrc.org).
Significant matches (E-value < 1e-5) from each genome were
obtained, and similarity scores for each significant match
were calculated as percent of the number of positive (i.e.,
identical plus conserved substitutions) amino acid residues
divided by the length of the query protein.
Acknowledgements
We are grateful to Graham Stewart for the clgR mutant and
complemented strain and Thomas Zahrt for the pepD mutant
and complemented strain. We also thank Karl Drlica, David
Perlin, Richard Pine and Xilin Zhao for critical comments on
the manuscript. The work was supported by NIH grants
GM096189 to OAI and HL 106788 to MLG. The authors have
no conflict of interest to declare.
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